Method for inline bilayer capacitance monitoring

ABSTRACT

A method of detecting a state of a lipid membrane in a cell of a nanopore based sequencing chip is disclosed. A lipid membrane is coupled with an integrating capacitor, wherein the lipid membrane is between a working electrode and a counter electrode. An alternating current (AC) voltage is applied to the counter electrode. A voltage across the integrating capacitor is periodically sampled by an analog-to-digital converter (ADC). A change in the sampled voltage across the integrating capacitor in response to an intermediate change in the AC voltage is determined. A state of the lipid membrane is determined based on the determined change in the sampled voltage across the integrating capacitor in response to the intermediate change in the AC voltage.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/842,654, filed Apr. 7, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/260,387, filed Jan. 29, 2019, now U.S. Pat. No.10,648,016, which is a continuation of U.S. patent application Ser. No.15/715,669, filed Sep. 26, 2017, titled “METHOD FOR INLINE BILAYERCAPACITANCE MONITORING”, which is a continuation-in-part of U.S. patentapplication Ser. No. 15/085,700 entitled NON-DESTRUCTIVE BILAYERMONITORING USING MEASUREMENT OF BILAYER RESPONSE TO ELECTRICAL STIMULUSfiled Mar. 30, 2016, now U.S. Pat. No. 10,155,979, each of which isherein incorporated by reference in its entirety for all purposes.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BACKGROUND

Advances in micro-miniaturization within the semiconductor industry inrecent years have enabled biotechnologists to begin packingtraditionally bulky sensing tools into smaller and smaller form factors,onto so-called biochips. Biochips may be used for nanopore-basedsequencing.

The step of inserting a nanopore into a lipid bilayer is performed afterit is determined that a lipid bilayer has been properly formed within acell of the nanopore based sequencing chip. In some techniques, theprocess of determining whether a lipid bilayer has been properly formedin a cell may cause an already properly formed lipid bilayer to bedestroyed. In other words, the stimulus voltage for testing the lipidbilayer may be destructive to the lipid bilayer. In the event that analready properly formed lipid bilayer is destroyed by the stimulusvoltage, a very high current begins to flow across the electrodes as aresult of the short-circuit condition. In response, the system may tryto re-form a new lipid bilayer in the particular cell again; however,this is both time-consuming and inefficient. In addition, the lipidbilayer may not re-form in the particular cell in a subsequent trial. Asa result, the overall percentage of cells in the nanopore basedsequencing chip with properly formed lipid bilayers and nanopores (i.e.,the yield of the nanopore based sequencing chip) is reduced. It would bedesirable to develop techniques for nanopore-based sequencing biochipsthat make them more robust, efficient, and cost-effective.

SUMMARY OF THE DISCLOSURE

In one aspect, the present invention provides methods of detecting astate of a lipid membrane in a cell of a nanopore based sequencing chip.In one embodiment, the method comprises the step of coupling a lipidmembrane with an integrating capacitor. In an additional embodiment, thelipid membrane is disposed between a working electrode and a counterelectrode. In another embodiment, the method further comprises the stepof applying an alternating current (AC) voltage to the counterelectrode. In one other embodiment, the method further comprises thestep of periodically sampling a voltage across the integrating capacitorby an analog-to-digital converter (ADC). In another embodiment, themethod further comprises the step of determining a change in the sampledvoltage across the integrating capacitor. In one additional embodiment,the determined change is in response to an intermediate change in the ACvoltage. In one embodiment, the method further comprises the step ofdetecting a state of the lipid membrane based on the determined changein the sampled voltage across the integrating capacitor. In oneadditional embodiment, the determined change is in response to theintermediate change in the AC voltage. In other embodiments, the step ofdetermining a change in the sampled voltage across the integratingcapacitor in response to the intermediate change in the AC voltagefurther comprises inserting the intermediate change in the AC voltagebetween two magnitudes of the AC voltage. In one other embodiment, themethod further comprises, in response to the detection that the lipidbilayer has ruptured, the step of disabling further electrical stimulifrom being applied across the lipid bilayer by opening a switch in thecell.

In one other embodiment, the method further comprises determining thechange in the sampled voltage across the integrating capacitor inresponse to the intermediate change in the AC voltage. In oneembodiment, the determining step is performed (i) when the AC voltage isswitching from a first phase to a second phase; or (ii) when the ACvoltage is switching from the second phase to the first phase. In onefurther embodiment, the AC voltage first phase comprises a first phasemagnitude and the AC voltage second phase comprises a second phasemagnitude. In one additional embodiment, the first phase magnitude ofthe AC voltage is greater than the second phase magnitude of the ACvoltage. In another embodiment, the first phase comprises a positivesquare wave and the second phase comprises a negative square wave. Inone other embodiment, the AC voltage is at an intermediate monitoringmagnitude, wherein the intermediate monitoring magnitude is smaller thanthe first phase magnitude but greater than the second phase magnitude.In one additional embodiment, the method further comprises the step ofselecting the intermediate monitoring magnitude based at least in parton an ADC reference window of the ADC.

In one embodiment, the method further comprises the step of comparingthe change in the sampled voltage across the integrating capacitoragainst one or more predetermined thresholds. In another embodiment, themethod further comprises the step of detecting the state of the lipidmembrane based on the comparisons against the one or more predeterminedthresholds. In one other embodiment, the state of the lipid membrane isselected from the group consisting of: a lipid membrane with more thantwo lipid molecule layers, a lipid bilayer, and a ruptured lipidbilayer. In a further embodiment, the method further comprises the stepof selecting the intermediate monitoring magnitude such that the sampledvoltage across the integrating capacitor is within an ADC referencewindow of the ADC in the event that the lipid bilayer has ruptured.

In one other aspect, the present invention provides methods thatcomprise determining the change in the sampled voltage across theintegrating capacitor in response to the intermediate change in the ACvoltage, wherein the cell is one of a plurality of cells in ananopore-based sequencing chip. In one embodiment, the determining stepis performed (i) when the AC voltage is switching from a first phase toa second phase; or (ii) when the AC voltage is switching from the secondphase to the first phase. In one further embodiment, the AC voltagefirst phase comprises a first phase magnitude and the AC voltage secondphase comprises a second phase magnitude. In one additional embodiment,the first phase magnitude of the AC voltage is greater than the secondphase magnitude of the AC voltage. In one other embodiment, the methodfurther comprises the step of pre-charging the integrating capacitor byconnecting the integrating capacitor to a constant pre-charging voltagesource using a global pre-charge signal. In another embodiment, theglobal pre-charge signal is used to control a timing of the pre-chargingof integrating capacitors in the plurality of cells. In one additionalembodiment, the method further comprises, after the integratingcapacitor is charged to the constant pre-charging voltage source value,the step of disconnecting the pre-charging voltage source from theintegrating capacitor using the global pre-charge signal. In one otherembodiment the global pre-charge signal is used to control a timing ofthe disconnecting of the pre-charging voltage source from integratingcapacitors in the plurality of cells. In a further embodiment, themethod further comprises the step of waiting a predetermined period oftime for the integrating capacitor to charge or discharge through acapacitance associated with the lipid bilayer. In one other embodiment,the method further comprises the step of sampling the voltage across theintegrating capacitor after the predetermined waiting period. In oneembodiment, the timing of the disconnecting of the pre-charging voltagesource from integrating capacitors in the plurality of cells isconfigured such that the timing is substantially the same as a timingwhen the AC voltage is switched to the intermediate monitoringmagnitude. In another embodiment, the timing of the pre-charging ofintegrating capacitors in the plurality of cells is configured such thatthe timing is after a frame of sequencing data from the plurality ofcells has been read out. In an additional embodiment, the frame is oneframe prior to a frame when the AC voltage is switched to theintermediate monitoring magnitude.

In one additional aspect, the present invention provides a system fordetecting a state of a lipid membrane in a cell of a nanopore basedsequencing chip. In one embodiment, the system comprises an integratingcapacitor. In another embodiment, the system further comprises a workingelectrode coupled to the integrating capacitor. In another embodiment,the system further comprises a counter electrode. In another embodiment,a lipid membrane is deposited (or disposed) between the workingelectrode and the counter electrode. In another embodiment, the lipidmembrane is coupled with the integrating capacitor. In anotherembodiment, the system further comprises an alternating current (AC)voltage source that applies an AC voltage to the counter electrode. Inanother embodiment, the system further comprises an analog-to-digitalconverter (ADC) periodically sampling a voltage across the integratingcapacitor. In another embodiment, the system further comprises aprocessor or a circuitry.

In another embodiment, the processor or circuitry is configured todetermine a change in the sampled voltage across the integratingcapacitor in response to an intermediate change in the AC voltage. In afurther embodiment, the processor or circuitry is further configured todetect a state of the lipid membrane based on the determined change inthe sampled voltage across the integrating capacitor in response to theintermediate change in the AC voltage. In an additional embodiment, theprocessor or circuitry is configured to determine a change in thesampled voltage across the integrating capacitor in response to theintermediate change in the AC voltage, wherein the determining comprisesinserting the intermediate change in the AC voltage between twomagnitudes of the AC voltage. In another embodiment, the processor orcircuitry is further configured to determine the change in the sampledvoltage across the integrating capacitor in response to the intermediatechange in the AC voltage when the AC voltage is switching from a firstphase to a second phase or when the AC voltage is switching from thesecond phase to the first phase. In one further embodiment, the ACvoltage first phase comprises a first phase magnitude and the AC voltagesecond phase comprises a second phase magnitude. In one additionalembodiment, the first phase magnitude of the AC voltage is greater thanthe second phase magnitude of the AC voltage. In another embodiment, thefirst phase comprises a positive square wave and the second phasecomprises a negative square wave. In an additional embodiment, theprocessor or circuitry is further configured to determine the change inthe sampled voltage across the integrating capacitor in response to theintermediate change in the AC voltage when the AC voltage is at anintermediate monitoring magnitude. In one other embodiment, theintermediate monitoring magnitude is smaller than the first phasemagnitude but greater than the second phase magnitude. In anotherembodiment, the processor or circuitry is further configured to selectthe intermediate monitoring magnitude based at least in part on a ADCreference window of the ADC. In an additional embodiment, the processoror circuitry is further configured to compare the change in the sampledvoltage across the integrating capacitor against one or morepredetermined thresholds. In one embodiment, the processor or circuitryis further configured to detect the state of the lipid membrane based onthe comparisons against the one or more predetermined thresholds. In oneadditional embodiment, the state of the lipid membrane is selected fromthe group consisting of: a lipid membrane with more than two lipidmolecule layers, a lipid bilayer, and a ruptured lipid bilayer.

In another aspect, the present invention provides a system for detectinga state of a lipid membrane in a cell of a nanopore based sequencingchip, wherein the system comprises a processor or circuitry configuredto determine the change in the sampled voltage across the integratingcapacitor in response to the intermediate change in the AC voltage,wherein the cell is one of a plurality of cells in a nanopore-basedsequencing chip. In one embodiment, the intermediate monitoringmagnitude is smaller than the first phase magnitude but greater than thesecond phase magnitude. In another embodiment, the system furthercomprises a constant pre-charging voltage source. In one additionalembodiment, the processor or circuitry is further configured topre-charge the integrating capacitor by connecting the integratingcapacitor to the constant pre-charging voltage source using a globalpre-charge signal. In one embodiment, the global pre-charge signal isused to control a timing of the pre-charging of integrating capacitorsin the plurality of cells. In another embodiment, the processor orcircuitry is further configured, after the integrating capacitor ischarged to the constant pre-charging voltage source value, to disconnectthe pre-charging voltage source from the integrating capacitor using theglobal pre-charge signal. In a further embodiment, the global pre-chargesignal is used to control a timing of the disconnecting of thepre-charging voltage source from integrating capacitors in the pluralityof cells. In an additional embodiment, the processor or circuitry isfurther configured to wait a predetermined period of time for theintegrating capacitor to charge or discharge through a capacitanceassociated with the lipid bilayer. In one other embodiment, theprocessor or circuitry is further configured to cause the ADC to samplethe voltage across the integrating capacitor after the predeterminedwaiting period. In one additional embodiment, the timing of thedisconnecting of the pre-charging voltage source from integratingcapacitors in the plurality of cells is configured such that the timingis substantially the same as a timing when the AC voltage is switched tothe intermediate monitoring magnitude. In one embodiment, the timing ofthe pre-charging of integrating capacitors in the plurality of cells isconfigured such that the timing is after a frame of sequencing data fromthe plurality of cells has been read out. In another embodiment, theframe is one frame prior to a frame when the AC voltage is switched tothe intermediate monitoring magnitude.

In a further embodiment, the system further comprises a switch in thecell controlled by the processor or the circuitry. In one embodiment,the processor or circuitry is further configured, in response to thedetection that the lipid bilayer has ruptured, to disable furtherelectrical stimulus from being applied across the lipid bilayer byopening the switch in the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip.

FIG. 2 illustrates an embodiment of a cell 200 performing nucleotidesequencing with the Nano-SBS technique.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags.

FIG. 5 illustrates an embodiment of a cell 500 in a nanopore basedsequencing chip.

FIG. 6A illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip, wherein the circuitry can be configuredto detect whether a lipid bilayer is formed in the cell without causingan already formed lipid bilayer to break down.

FIG. 6B illustrates the same circuitry 600 in a cell of a nanopore basedsequencing chip as that shown in FIG. 6A. In comparison with FIG. 6A,instead of showing a lipid membrane/bilayer between the workingelectrode and the counter electrode, an electrical model representingthe electrical properties of the working electrode and the lipidmembrane/bilayer is shown.

FIG. 7 illustrates an electrical model 700 representing the electricalproperties of a portion of circuitry 600 during the lipid bilayermeasurement phase of the system.

FIG. 8A illustrates that a small observed positive/negative voltagechange ±ΔV_(ADC) in response to a positive/negative voltage change±ΔV_(liq) detects that no lipid bilayer has been formed in the cell.

FIG. 8B illustrates that a large observed positive/negative voltagechange ±ΔV_(ADC) in response to a positive/negative voltage change±ΔV_(liq) detects that a lipid bilayer has been formed in a cell.

FIG. 9A illustrates an exemplary plot of V_(ADC) versus time before andafter a lipid bilayer is formed within a cell.

FIG. 9B illustrates a zoomed-in view of the exemplary plot of V_(ADC)versus time (see FIG. 9A) during the time period t₁ when a lipid bilayerhas not been formed.

FIG. 9C illustrates a zoomed-in view of the exemplary plot of V_(ADC)versus time (see FIG. 9A) during the time period t₂ when a lipid bilayerhas been formed.

FIG. 10 illustrates an electrical model 1000 representing the electricalproperties of a portion of circuitry 600 during the sequencing phase ofthe system.

FIG. 11A illustrates that a large observed positive/negative voltagechange ±ΔV_(ADC) in response to a positive/negative voltage change±ΔV_(liq) indicates that a lipid bilayer is still intact in a cell. FIG.11A is identical to FIG. 8B.

FIG. 11B illustrates that a further increase in the observedpositive/negative voltage change ±ΔV_(ADC) in response to apositive/negative voltage change ±ΔV_(liq) indicates that a lipidbilayer has just been ruptured in the cell.

FIG. 12 illustrates an exemplary plot of an improved V_(liq) waveform1200 for detecting a state of a lipid membrane in a cell of a nanoporebased sequencing chip during different phases (including the sequencingphase) of the system.

FIG. 13 illustrates that to detect a cell with a short-circuit conditionduring the sequencing phase, circuitry 600 may monitor a delta voltagechange, ΔV_(ADC), at integrating capacitor 608 (neap) in response to adelta voltage change (ΔV_(liq)) applied to the bulk liquid in contactwith the lipid membrane/bilayer when Vliq switches from a brightmagnitude to a monitoring signal magnitude.

FIG. 14 illustrates that the cells in any given row of a cell bank sharethe same integration time intervals, but that the integration timeintervals of adjacent rows are staggered from each other, causing a rowdependence effect which degrades the overall performance of thedetection of ruptured lipid bilayers.

FIG. 15 illustrates that a global pre-charge signal 1502 is used tosynchronize the integration of the cells in different rows of the cellbank when V_(liq) is about to switch to the intermediate monitoringsignal.

FIG. 16 illustrates that a modified global pre-charge signal 1602 isused to synchronize the integration of the cells in different rows ofthe cell bank when V_(liq) is about to switch to the intermediatemonitoring signal.

FIG. 17A illustrates the row dependence effect of the ruptured lipidbilayer detection technique.

FIG. 17B illustrates that the row dependence effect is significantlyreduced by using the global pre-charge signal in FIG. 16 .

FIG. 18A is a plot of the observed voltage change ΔV_(ADC) in responseto the monitoring signal in cells that belong to different rows of acell bank when ruptured lipid bilayers are detected.

FIG. 18B is a histogram that shows the distribution of the responsesignals when ruptured lipid bilayers are detected.

FIG. 18C is a plot of the observed voltage change ΔV_(ADC) in responseto the monitoring signal in cells that belong to different rows of acell bank when ruptured lipid bilayers are not detected.

FIG. 18D is a histogram that shows the distribution of the responsesignals when ruptured lipid bilayers are not detected.

FIG. 19 illustrates an embodiment of a process 1900 for an improvedtechnique of forming lipid layers in the cells of a nanopore basedsequencing chip.

FIG. 20 illustrates the top view of a nanopore based sequencing system2000 with an improved flow chamber enclosing a silicon chip that allowsliquids and gases to pass over and contact sensors on the chip surface.

FIG. 21A illustrates the initial distribution of cells with differentΔV_(ADC) values.

FIG. 21B illustrates the distribution of cells with different ΔV_(ADC)values after the lipid-thinning stimulus phase and salt buffer solutionflowing phase of process 1000 have repeated a number of times.

FIG. 21C illustrates the distribution of cells with different ΔV_(ADC)values after the lipid-thinning stimulus phase and salt buffer solutionflowing phase of process 1000 have repeated an even greater number oftimes.

FIG. 22 illustrates an embodiment of a timing diagram for a bilayermeasurement phase, an electrical lipid-thinning stimulus phase, and asalt buffer solution flowing phase.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims andthe invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

Nanopore membrane devices having pore sizes on the order of onenanometer in internal diameter have shown promise in rapid nucleotidesequencing. When a voltage potential is applied across a nanoporeimmersed in a conducting fluid, a small ion current attributed to theconduction of ions across the nanopore can be observed. The size of thecurrent is sensitive to the pore size.

A nanopore based sequencing chip may be used for DNA sequencing. Ananopore based sequencing chip incorporates a large number of sensorcells configured as an array. For example, an array of one million cellsmay include 1000 rows by 1000 columns of cells.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip. A membrane 102 is formed over the surface of the cell.In some embodiments, membrane 102 is a lipid bilayer. The bulkelectrolyte 114 containing soluble protein nanopore transmembranemolecular complexes (PNTMC) and the analyte of interest is placeddirectly onto the surface of the cell. A single PNTMC 104 is insertedinto membrane 102 by electroporation. The individual membranes in thearray are neither chemically nor electrically connected to each other.Thus, each cell in the array is an independent sequencing machine,producing data unique to the single polymer molecule associated with thePNTMC. PNTMC 104 operates on the analytes and modulates the ioniccurrent through the otherwise impermeable bilayer.

With continued reference to FIG. 1 , analog measurement circuitry 112 isconnected to a metal electrode 110 covered by a thin film of electrolyte108. The thin film of electrolyte 108 is isolated from the bulkelectrolyte 114 by the ion-impermeable membrane 102. PNTMC 104 crossesmembrane 102 and provides the only path for ionic current to flow fromthe bulk liquid to working electrode 110. The cell also includes acounter electrode (CE) 116, which is an electrochemical potentialsensor. The cell also includes a reference electrode 117.

In some embodiments, a nanopore array enables parallel sequencing usingthe single molecule nanopore-based sequencing by synthesis (Nano-SBS)technique. FIG. 2 illustrates an embodiment of a cell 200 performingnucleotide sequencing with the Nano-SBS technique. In the Nano-SBStechnique, a template 202 to be sequenced and a primer are introduced tocell 200. To this template-primer complex, four differently taggednucleotides 208 are added to the bulk aqueous phase. As the correctlytagged nucleotide is complexed with the polymerase 204, the tail of thetag is positioned in the barrel of nanopore 206. The tag held in thebarrel of nanopore 206 generates a unique ionic blockade signal 210,thereby electronically identifying the added base due to the tags'distinct chemical structures.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags. A nanopore 301 is formed in a membrane302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) isassociated with the nanopore. In some cases, polymerase 303 iscovalently attached to nanopore 301. Polymerase 303 is associated with anucleic acid molecule 304 to be sequenced. In some embodiments, thenucleic acid molecule 304 is circular. In some cases, nucleic acidmolecule 304 is linear. In some embodiments, a nucleic acid primer 305is hybridized to a portion of nucleic acid molecule 304. Polymerase 303catalyzes the incorporation of nucleotides 306 onto primer 305 usingsingle stranded nucleic acid molecule 304 as a template. Nucleotides 306comprise tag species (“tags”) 307.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags. Stage A illustrates the components asdescribed in FIG. 3 . Stage C shows the tag loaded into the nanopore. A“loaded” tag may be one that is positioned in and/or remains in or nearthe nanopore for an appreciable amount of time, e.g., 0.1 millisecond(ms) to 10000 ms. In some cases, a tag that is pre-loaded is loaded inthe nanopore prior to being released from the nucleotide. In someinstances, a tag is pre-loaded if the probability of the tag passingthrough (and/or being detected by) the nanopore after being releasedupon a nucleotide incorporation event is suitably high, e.g., 90% to99%.

At stage A, a tagged nucleotide (one of four different types: A, T, G,or C) is not associated with the polymerase. At stage B, a taggednucleotide is associated with the polymerase. At stage C, the polymeraseis docked to the nanopore. The tag is pulled into the nanopore duringdocking by an electrical force, such as a force generated in thepresence of an electric field generated by a voltage applied across themembrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with thenucleic acid molecule. These non-paired nucleotides typically arerejected by the polymerase within a time scale that is shorter than thetime scale for which correctly paired nucleotides remain associated withthe polymerase. Since the non-paired nucleotides are only transientlyassociated with the polymerase, process 400 as shown in FIG. 4 typicallydoes not proceed beyond stage D. For example, a non-paired nucleotide isrejected by the polymerase at stage B or shortly after the processenters stage C.

Before the polymerase is docked to the nanopore, the conductance of thenanopore is ˜300 picosiemens (300 pS). At stage C, the conductance ofthe nanopore is about 60 pS, 80 pS, 100 pS, or 120 pS, corresponding toone of the four types of tagged nucleotides respectively. The polymeraseundergoes an isomerization and a transphosphorylation reaction toincorporate the nucleotide into the growing nucleic acid molecule andrelease the tag molecule. In particular, as the tag is held in thenanopore, a unique conductance signal (e.g., see signal 210 in FIG. 2 )is generated due to the tag's distinct chemical structures, therebyidentifying the added base electronically. Repeating the cycle (i.e.,stage A through E or stage A through F) allows for the sequencing of thenucleic acid molecule. At stage D, the released tag passes through thenanopore.

In some cases, tagged nucleotides that are not incorporated into thegrowing nucleic acid molecule will also pass through the nanopore, asseen in stage F of FIG. 4 . The unincorporated nucleotide can bedetected by the nanopore in some instances, but the method provides ameans for distinguishing between an incorporated nucleotide and anunincorporated nucleotide based at least in part on the time for whichthe nucleotide is detected in the nanopore. Tags bound to unincorporatednucleotides pass through the nanopore quickly and are detected for ashort period of time (e.g., less than 10 ms), while tags bound toincorporated nucleotides are loaded into the nanopore and detected for along period of time (e.g., at least 10 ms).

FIG. 5 illustrates an embodiment of a cell 500 in a nanopore basedsequencing chip. Cell 500 includes a dielectric layer 501. Dielectricmaterial used to form dielectric layer 501 includes glass, oxides,nitrides, and the like. Cell 500 further includes a dielectric layer 504above dielectric layer 501. Dielectric layer 504 forms the wallssurrounding a well 505 in which a working electrode 502 is located atthe bottom. Dielectric material used to form dielectric layer 504includes glass, oxide, silicon mononitride (SiN), and the like. The topsurface of dielectric layer 504 may be silanized. Silanization forms ahydrophobic layer 520 above the top surface of dielectric layer 504. Insome embodiments, hydrophobic layer 520 has a thickness of about 1.5nanometer (nm).

Well 505 formed by the dielectric layer walls 504 further includes afilm of salt solution 506 above working electrode 502. Salt solution 506may include one of the following: lithium chloride (LiCl), sodiumchloride (NaCl), potassium chloride (KCl), lithium glutamate, sodiumglutamate, potassium glutamate, lithium acetate, sodium acetate,potassium acetate, calcium chloride (CaCl₂)), strontium chloride(SrCl₂), Manganese chloride (MnCl₂), and magnesium chloride (MgCl₂). Insome embodiments, the film of salt solution 506 has a thickness of aboutthree microns (μm).

As shown in FIG. 5 , a membrane is formed on top of dielectric layer 504and spans across well 505. For example, the membrane includes a lipidmonolayer 518 formed on top of hydrophobic layer 520. As the membranereaches the opening of well 505, the lipid monolayer transitions to alipid bilayer 514 that spans across the opening of the well. A bulkelectrolyte 508 containing protein nanopore transmembrane molecularcomplexes (PNTMC) and the analyte of interest is placed directly abovethe well. A single PNTMC/nanopore 516 is inserted into lipid bilayer 514by electroporation. Nanopore 516 crosses lipid bilayer 514 and providesthe only path for ionic flow from bulk electrolyte 508 to workingelectrode 502. Bulk electrolyte 508 may further include one of thefollowing: lithium chloride (LiCl), sodium chloride (NaCl), potassiumchloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂)), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂).

Cell 500 includes a counter electrode (CE) 510, which is anelectrochemical potential sensor. Cell 500 also includes a referenceelectrode 512. In some embodiments, counter electrode 510 is sharedbetween a plurality of cells, and is therefore also referred to as acommon electrode. The common electrode can be configured to apply acommon potential to the bulk liquid in contact with the nanopores in themeasurements cells. The common potential and the common electrode arecommon to all of the measurement cells.

In some embodiments, working electrode 502 is a metal electrode. Fornon-faradaic conduction, working electrode 502 may be made of metalsthat are resistant to corrosion and oxidation, e.g., platinum, gold,titanium nitride and graphite. For example, working electrode 502 may bea platinum electrode with electroplated platinum. In another example,working electrode 502 may be a titanium nitride (TiN) working electrode.

The step of inserting a nanopore into a lipid bilayer is performed afterit is determined that a lipid bilayer has been properly formed within acell of the nanopore based sequencing chip. In some techniques, theprocess of determining whether a lipid bilayer has been properly formedin a cell may cause an already properly formed lipid bilayer to bedestroyed. For example, a stimulus voltage may be applied to cause acurrent to flow across the electrodes. Although the measured response tothe stimulus voltage may be used to distinguish between a cell with aproperly formed lipid bilayer (i.e., a lipid bilayer that is two layersof lipid molecules thick) from a cell without a properly formed lipidbilayer (e.g., a cell with a thick lipid and solvent combined film thatspans across the well of the cell), the stimulus voltage level is highenough to cause an already properly formed lipid bilayer to break downin some instances. In other words, the stimulus voltage for testing thelipid bilayer may be destructive to the lipid bilayer. In the event thatan already properly formed lipid bilayer is destroyed by the stimulusvoltage, a very high current begins to flow across the electrodes as aresult of the short-circuit condition. In response, the system may tryto reform a new lipid bilayer in the particular cell again; however,this is both time-consuming and inefficient. In addition, a lipidbilayer may not reform in the particular cell in a subsequent trial. Asa result, the overall percentage of cells in the nanopore basedsequencing chip with properly formed lipid bilayers and nanopores (i.e.,the yield of the nanopore based sequencing chip) is reduced.

A non-destructive technique to detect a lipid bilayer formed in a cellof a nanopore based sequencing chip is disclosed. A non-destructivetechnique to detect a lipid bilayer has many advantages, includingincreasing the efficiency and yield of the nanopore based sequencingchip.

FIG. 6A illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip wherein the circuitry can be configuredto detect whether a lipid bilayer is formed in the cell without causingan already formed lipid bilayer to break down.

FIG. 6A shows a lipid membrane or lipid bilayer 612 situated between acell working electrode 614 and a counter electrode 616, such that avoltage is applied across lipid membrane/bilayer 612. A lipid bilayer isa thin membrane made of two layers of lipid molecules. A lipid membraneis a membrane made of several layers (more than two) of lipid molecules.Lipid membrane/bilayer 612 is also in contact with a bulkliquid/electrolyte 618. Note that working electrode 614, lipidmembrane/bilayer 612, and counter electrode 616 are drawn upside down ascompared to the working electrode, lipid bilayer, and counter electrodein FIG. 1 . In some embodiments, the counter electrode is shared betweena plurality of cells, and is therefore also referred to as a commonelectrode. The common electrode can be configured to apply a commonpotential to the bulk liquid in contact with the lipidmembranes/bilayers in the measurements cells by connecting the commonelectrode to a voltage source V_(liq) 620. The common potential and thecommon electrode are common to all of the measurement cells. There is aworking cell electrode within each measurement cell; in contrast to thecommon electrode, working cell electrode 614 is configurable to apply adistinct potential that is independent from the working cell electrodesin other measurement cells.

FIG. 6B illustrates the same circuitry 600 in a cell of a nanopore basedsequencing chip as that shown in FIG. 6A. In comparison with FIG. 6A,instead of showing a lipid membrane/bilayer between the workingelectrode and the counter electrode, an electrical model representingthe electrical properties of the working electrode and the lipidmembrane/bilayer is shown.

Electrical model 622 includes a capacitor 624 representing theelectrical properties of working electrode 614. The capacitanceassociated with working electrode 614 is also referred to as a doublelayer capacitance (C_(double layer)). Electrical model 622 furtherincludes a capacitor 626(C_(bilayer)) that models a capacitanceassociated with the lipid membrane/bilayer and a resistor 628(R_(bilayer)) that models a resistance associated with the lipidmembrane/bilayer. The resistance associated with the lipidmembrane/bilayer is very high, and therefore R_(bilayer) may be replacedby an open circuit, which reduces electrical model 622 toC_(double layer) in series with C_(bilayer).

Voltage source V_(liq) 620 is an alternating current (AC) voltagesource. Counter electrode 616 is immersed in the bulk liquid 618, and anAC non-Faradaic mode is utilized to modulate a square wave voltageV_(liq) and apply it to the bulk liquid in contact with the lipidmembranes/bilayers in the measurement cells. In some embodiments,V_(liq) is a square wave with a magnitude of ±200-250 mV and a frequencybetween 25 and 100 Hz.

Pass device 606 is a switch that can be used to connect or disconnectthe lipid membrane/bilayer and the electrodes from the measurementcircuitry 600. The switch enables or disables a voltage stimulus thatcan be applied across the lipid membrane/bilayer in the cell. Beforelipids are deposited to the cell to form a lipid bilayer, the impedancebetween the two electrodes is very low because the well of the cell isnot sealed, and therefore switch 606 is kept open to avoid ashort-circuit condition. Switch 606 may be closed once lipid solvent hasbeen deposited to the cell that seals the well of the cell.

Circuitry 600 further includes an on-chip fabricated integratingcapacitor 608 (n_(cap)). Integrating capacitor 608 is pre-charged byusing a reset signal 603 to close switch 601, such that integratingcapacitor 608 is connected to a voltage source V_(pre) 605. In someembodiments, voltage source V_(pre) 605 provides a constant positivevoltage with a magnitude of 900 mV. When switch 601 is closed,integrating capacitor 608 is pre-charged to the positive voltage levelof voltage source V_(pre) 605.

After integrating capacitor 608 is pre-charged, reset signal 603 is usedto open switch 601 such that integrating capacitor 608 is disconnectedfrom voltage source V_(pre) 605. At this point, depending on the levelof V_(liq), the potential of counter electrode 616 may be at a higherlevel than the potential of working electrode 614, or vice versa. Forexample, during the positive phase of square wave V_(liq) (i.e., thedark period of the AC voltage source signal cycle), the potential ofcounter electrode 616 is at a higher level than the potential of workingelectrode 614. Similarly, during the negative phase of square waveV_(liq) (i.e., the bright period of the AC voltage source signal cycle),the potential of counter electrode 616 is at a lower level than thepotential of working electrode 614. Due to this potential difference,integrating capacitor 608 may be charged during the dark period of theAC voltage source signal cycle and discharged during the bright periodof the AC voltage source signal cycle.

Depending on the sampling rate of an analog-to-digital converter (ADC)610, integrating capacitor 608 charges or discharges for a fixed periodof time, and then the voltage stored in integrating capacitor 608 may beread out by ADC 610. After the sampling by ADC 610, integratingcapacitor 608 is pre-charged again by using reset signal 603 to closeswitch 601, such that integrating capacitor 608 is connected to voltagesource V_(pre) 605 again. In some embodiments, the sampling rate of ADC610 is between 1500 to 2000 Hz. In some embodiments, the sampling rateof ADC 610 is up to 5 kHz. For example, with a sampling rate of 1 kHz,integrating capacitor 608 charges or discharges for a period of −1 ms,and then the voltage stored in integrating capacitor 608 is read out byADC 610. After the sampling by ADC 610, integrating capacitor 608 ispre-charged again by using reset signal 603 to close switch 601 suchthat integrating capacitor 608 is connected to voltage source V_(pre)605 again. The steps of pre-charging the integrating capacitor 608,waiting a fixed period of time for the integrating capacitor 608 tocharge or discharge, and sampling the voltage stored in integratingcapacitor by ADC 610 are then repeated in cycles throughout a lipidbilayer measurement phase of the system.

Circuitry 600 may be used to detect whether a lipid bilayer is formed inthe cell by monitoring a delta voltage change, ΔV_(ADC), at integratingcapacitor 608 (n_(cap)) in response to a delta voltage change (ΔV_(liq))applied to the bulk liquid in contact with the lipid membrane/bilayer.As will be described in greater detail below, during the lipid bilayermeasurement phase, circuitry 600 may be modeled as a voltage dividerwith C_(bilayer) 626, C_(double layer) 624, and n_(cap) 608 connected inseries, and a voltage change tapped at an intermediate point of thevoltage divider can be read by ADC 610 for determining whether a lipidbilayer has been formed.

FIG. 7 illustrates an electrical model 700 representing the electricalproperties of a portion of circuitry 600 during the lipid bilayermeasurement phase of the system. As shown in FIG. 7 , C_(double layer)624 is connected in series with C_(bilayer) 626, but R_(bilayer) 628(see FIG. 6B) is eliminated from electrical model 700. R_(bilayer) 628can be removed from electrical model 700 because the resistanceassociated with the lipid membrane/bilayer is very high, and thereforeR_(bilayer) may be approximated as an open circuit. As shown in FIG. 7 ,C_(double layer) 624 and C_(bilayer) 626 are further connected in serieswith n_(cap) 608.

When operating in an AC mode, the voltage read by the ADC (V_(ADC)) canbe determined by:

$\begin{matrix}{V_{ADC} = {V_{liq}*\frac{Z({ncap})}{{Z({bilayer})} + {Z\left( {{double}{layer}} \right)} + {Z({ncap})}}}} & \left( {{Equation}1} \right)\end{matrix}$ whereZ = 1/(jωC),Z(ncap)istheACimpedanceassociatedwithn_(cap),Z(doublelayer)istheACimpedanceassociatedwiththeworkingelectrode,andZ(bilayer)istheACimpedanceassociatedwiththelipidmembraned/bilayer

The AC impedance of the double layer, Z(double layer), has a very lowvalue compared to Z(bilayer) and Z(ncap) because C_(double layer) ismuch larger than C_(bilayer) or the capacitance of n_(cap). Therefore,substituting Z(ncap)=1/(jωCn_(cap)), Z (bilayer)=1/jωC_(bilayer), andZ(double layer)=0, equation (1) can be simplified as:

$\begin{matrix}{V_{ADC} = {V_{liq}*\frac{C({bilayer})}{{C({ncap})} + {C({bilayer})}}}} & {{Equation}(2)}\end{matrix}$ whereC(ncap)isthecapacitanceassociatedwithn_(cap),andC(bilayer)isthecapacitanceassociatedwiththelipidmembrane/bilayer

When lipids are first deposited into the cells to form the lipidbilayers, some of the cells have lipid bilayers spontaneously formed,but some of the cells merely have a thick lipid membrane (with multiplelayers of lipid molecules and solvent combined together) spanning acrosseach of the wells of the cells. The capacitance associated with a lipidbilayer is larger than the capacitance associated with a lipid membranethat is more than two layers of lipid molecules thick because thecapacitance of the lipid membrane/bilayer is inversely proportional toits thickness. As a lipid membrane thins out and transitions to become alipid bilayer, the thickness decreases and its associated capacitanceincreases. In Equation (2) above, as a lipid bilayer begins to formwithin a cell, C(bilayer) increases while C(ncap) remains constant, suchthat on the whole V_(ADC) increases. An increase in V_(ADC) cantherefore be used as an indicator that a lipid bilayer has been formedwithin a cell.

In some embodiments, a delta voltage change ΔV_(ADC) at integratingcapacitor 608 (neap) in response to a delta voltage change (ΔV_(liq))applied to the bulk liquid in contact with the lipid membrane/bilayer ismonitored in order to detect whether a lipid bilayer has been formed ina cell. For example, Equation (2) may be rewritten as:

$\begin{matrix}{{\Delta V_{ADC}} = {\Delta V_{liq}*\frac{C({bilayer})}{{C({ncap})} + {C({bilayer})}}}} & {{Equation}(3)}\end{matrix}$whereΔV_(ADC)isavoltagechangeatintegratingcapacitor608(n_(cap))readbytheADC,ΔV_(liq)isavoltagechangeappliedtothebulkliquid,C(ncap)isthecapacitanceassociatedwithn_(cap),andC(bilayer)isthecapacitanceassociatedwiththelipidmembrane/bilayer.

In Equation (3) above, because C(ncap) remains constant, whileC(bilayer) increases as a lipid bilayer begins to form within a cell,ΔV_(ADC) increases as well. ΔV_(ADC) is roughly proportional to thecapacitance associated with the lipid membrane/bilayer, C(bilayer). Anincrease in ΔV_(ADC) can therefore be used as an indicator that a lipidbilayer has been formed within a cell.

In some embodiments, in order to maximize the observable ΔV_(ADC) for amore reliable detection of a lipid bilayer, ΔV_(ADC) in response to amaximum voltage change applied to the bulk liquid in contact with thelipid membrane/bilayer (max ΔV_(liq)) is monitored in order to detectwhether a lipid bilayer has been formed in a cell.

FIG. 8A illustrates that a small observed positive/negative voltagechange ±ΔV_(ADC) in response to a positive/negative voltage change±ΔV_(liq) results in no lipid bilayer being detected to have been formedin the cell. FIG. 8B illustrates that a large observed positive/negativevoltage change ±ΔV_(ADC) in response to a positive/negative voltagechange ±ΔV_(liq) results in the detection of a lipid bilayer having beenformed in a cell.

In FIG. 8A, a maximum positive voltage change +ΔV_(liq) occurs when thesquare wave V_(liq) changes from a negative phase to a positive phase,while a maximum negative voltage change −ΔV_(liq) occurs when the squarewave V_(liq) changes from a positive phase to a negative phase. In FIG.8A, at the instance when ΔVliq is at a positive maximum, only a small+ΔV_(ADC) can be observed if a lipid bilayer has not been formed in thecell; at the instance when ΔV_(liq) is at a negative maximum, only asmall —ΔV_(ADC) can be observed if a lipid bilayer has not been formedin the cell.

In FIG. 8B, at the instance when ΔV_(liq) is at a positive maximum, alarge positive voltage change +ΔV_(ADC) can be observed if a lipidbilayer has already been formed in the cell. And at the instance whenΔV_(liq) is at a negative maximum, a large negative voltage change−ΔV_(ADC) can be observed if a lipid bilayer has already been formed inthe cell.

In some embodiments, the absolute value of ΔV_(ADC) (|ΔV_(ADC)|)observed when the absolute value of ΔV_(liq) (|ΔV_(liq)|) is at amaximum is compared with a predetermined threshold. If(|ΔV_(ADC)>predetermined threshold), then it is determined that a lipidbilayer is detected. Conversely, if (|ΔV_(ADC)|<predeterminedthreshold), then it is determined that a lipid bilayer is not detected.

FIG. 9A illustrates an exemplary plot of V_(ADC) versus time before andafter a lipid bilayer is formed within a cell. The plot in FIG. 9A isbased on real testing data. As shown in FIG. 9A, the units of V_(ADC) onthe y-axis are in ADC counts. However, other units may be used as well.As shown in FIG. 9A, during a time period t1 when a lipid bilayer hasnot been formed, the recorded |ΔV_(ADC)| values are smaller than thoserecorded during a time period t2 after a lipid bilayer has been formedin the cell.

FIG. 9B illustrates a zoomed-in view of the exemplary plot of V_(ADC)versus time (see FIG. 9A) during the time period t1 when a lipid bilayerhas not been formed. The results shown in FIG. 9B are consistent withFIG. 8A. In FIG. 9B, a maximum +ΔV_(liq) occurs when the square waveV_(liq) changes from a negative phase to a positive phase, while amaximum −ΔV_(liq) occurs when the square wave V_(liq) changes from apositive phase to a negative phase. In FIG. 9B, at the instance whenΔV_(liq) is at a positive maximum, only a small +ΔV_(ADC) can beobserved because a lipid bilayer has not been formed in the cell; at theinstance when ΔV_(liq) is at a negative maximum, only a small —ΔV_(ADC)can be observed because a lipid bilayer has not been formed in the cell.

FIG. 9C illustrates a zoomed-in view of the exemplary plot of V_(ADC)versus time (see FIG. 9A) during the time period t2 when a lipid bilayerhas been formed. The results shown in FIG. 9C are consistent with FIG.8B. In FIG. 9C, at the instance when ΔV_(liq) is at a positive maximum,a large +ΔV_(ADC) can be observed between two consecutive sample pointsbecause a lipid bilayer has already been formed in the cell. At theinstance when ΔV_(liq) is at a negative maximum, a large −ΔV_(ADC) canbe observed because a lipid bilayer has already been formed in the cell.Note that shortly after the square wave V_(liq) changes from one phaseto another, ΔV_(liq) stays at zero, and V_(ADC) reduces to zero inresponse. As shown in FIG. 9C, when a lipid bilayer has already beenformed in the cell, a positive or negative spike in V_(ADC) can beobserved. The positive or negative spikes are followed by much smallerV_(ADC) values.

After it is determined that a lipid bilayer has been properly formedwithin a cell of the nanopore based sequencing chip using the abovedescribed technique, a nanopore may be inserted into the lipid bilayer,and the cell with the inserted nanopore may be used for nucleic acidsequencing. During the sequencing phase, the lipid bilayers in some ofthe cells in the nanopore based sequencing chip may rupture due toosmotic imbalance or other reasons. A ruptured lipid bilayer in a cellis undesirable because it causes a very high current to flow across theelectrodes as a result of a short-circuit like condition, and the highcurrent may also affect data acquisition in neighboring cells.Therefore, a technique for detecting during the sequencing phase a cellthat has a short-circuit condition due to a ruptured lipid bilayer isdesirable. The technique enables the nanopore based sequencing chip todisable the detected cell, thereby improving the overall performance ofthe chip.

Disabling a cell with a short-circuit condition may be achieved bycontrolling pass device 606 as shown in FIG. 6A or FIG. 6B. Pass device606 may be used as a switch to disable the cell by disconnecting theelectrodes from the measurement circuitry 600, such that a voltagestimulus is no longer applied across the ruptured lipid bilayer/membranein the cell.

Detecting a cell with a short-circuit condition during the sequencingphase may be achieved by using circuitry 600 to monitor a delta voltagechange, ΔV_(ADC), at integrating capacitor 608 (n_(cap)) in response toa delta voltage change (ΔV_(liq)) applied to the bulk liquid in contactwith the lipid membrane/bilayer, which is similar to the techniquedescribed above for detecting the formation of a lipid bilayer in acell. As will be described in greater detail below, during thesequencing phase, circuitry 600 may be modeled as a voltage divider withncap connected in series with an impedance Z 1002 associated with theworking electrode and the lipid bilayer/membrane, which is similar tomodel 7000 described earlier for modeling circuitry 600 during the lipidbilayer measurement phase. A voltage change tapped at an intermediatepoint of the voltage divider can be read by ADC 610 for determiningwhether there is a short-circuit condition due to a ruptured lipidbilayer in the cell.

FIG. 10 illustrates an electrical model 1000 representing the electricalproperties of a portion of circuitry 600 during the sequencing phase ofthe system. As shown in FIG. 10 , an impedance Z 1002 is used to modelthe working electrode and lipid bilayer/membrane, and Z 1002 isconnected in series with n_(cap) 608.

When operating in an AC mode, the voltage read by the ADC (V_(ADC)) canbe determined by:

$\begin{matrix}{V_{ADC} = {V_{liq}*\frac{Z({ncap})}{{Z1002} + {Z({ncap})}}}} & {{Equation}(4)}\end{matrix}$ whereZ = 1/(jωC),Z(ncap)istheACimpedanceassociatedwithn_(cap),andZ1002istheACimpedanceassociatedwiththeworkingelectrodeandthelipidbilayer/membrane.

In Equation (4) above, as the lipid bilayer ruptures, Z 1002 decreasessignificantly because of the short-circuited condition, such thatV_(ADC) has a value that is close to V_(liq). A further increase inV_(ADC) can therefore be used as an indicator that a lipid bilayer hasbeen ruptured within a cell.

In some embodiments, a delta voltage change ΔV_(ADC) at integratingcapacitor 608 (n_(cap)) in response to a delta voltage change (ΔV_(liq))applied to the bulk liquid in contact with the lipid membrane/bilayer ismonitored in order to detect whether a lipid bilayer has been rupturedin a cell. For example, Equation (4) may be rewritten as:

$\begin{matrix}{{\Delta V_{ADC}} = {\Delta V_{liq}*\frac{Z({ncap})}{{Z1002} + {Z({ncap})}}}} & {{Equation}(3)}\end{matrix}$whereΔV_(ADC)isavoltagechangeatintegratingcapacitor608(n_(cap))readbytheADC,ΔV_(liq)isavoltagechangeappliedtothebulkliquid,Z(ncap)istheACimpedanceassociatedwithn_(cap),andZ1002istheACimpedanceassociatedwithworkingelectrodeandthelipidbilayer/membrane.

In Equation (5) above, because Z 1002 decreases as the lipid bilayerruptures within a cell, ΔV_(ADC) increases to a value that is close toΔV_(liq). A further increase in ΔV_(ADC) can therefore be used as anindicator that a lipid bilayer has just been ruptured within a cell.

In some embodiments, in order to maximize the observable ΔV_(ADC) for amore reliable detection of a ruptured lipid bilayer, ΔV_(ADC) inresponse to a maximum voltage change applied to the bulk liquid incontact with the lipid membrane/bilayer (max ΔV_(liq)) is monitored inorder to detect whether a lipid bilayer has just been ruptured in acell.

FIG. 11A illustrates that a large observed positive/negative voltagechange ±ΔV_(ADC) in response to a positive/negative voltage change±ΔV_(liq) indicates that a lipid bilayer is still intact in a cell. FIG.11A is identical to FIG. 8B. FIG. 11B illustrates that a furtherincrease in the observed positive/negative voltage change ±ΔV_(ADC) inresponse to a positive/negative voltage change ±ΔV_(liq) indicates thata lipid bilayer has just been ruptured in the cell.

In FIG. 11B, at the instance when ΔV_(liq) is at a positive maximum, afurther increase in magnitude of a positive voltage change +ΔV_(ADC) canbe observed if a lipid bilayer has just been ruptured in the cell. Andat the instance when ΔV_(liq) is at a negative maximum, a furtherincrease in magnitude of a negative voltage change —ΔV_(ADC) can beobserved if a lipid bilayer has just been ruptured in the cell.

In view of FIGS. 8A, 8B, 11A, and 11B, the absolute value of ΔV_(ADC)(|ΔV_(ADC)|) observed when the absolute value of ΔV_(liq) (|ΔV_(liq)|)is at a maximum may be compared with one or more predeterminedthresholds in order to determine the state of a lipid membrane. Forexample, two threshold levels may be used to determine the state of alipid membrane, where threshold1<threshold2. If (|ΔV_(ADC)|<threshold1),then it is determined that the lipid membrane has multiple layers oflipid molecules and solvent combined together, and is not yet a lipidbilayer. If ((threshold1<=|ΔV_(ADC)|<threshold2), then it is determinedthat a lipid bilayer is formed. If (|ΔV_(ADC)|>=threshold2), then it isdetermined that a lipid bilayer is ruptured.

However, applying the above described diagnostic technique to detect aruptured lipid bilayer in a cell of a nanopore based sequencing chipduring the sequencing phase of the system has a number of challenges.One of the challenges is selecting a suitable ADC reference window and asuitable V_(liq) magnitude that work well for both nucleic acidsequencing (the main experiment) and the diagnostic test. An ADCreference window is the range of minimum to maximum analog values thatthe ADC can convert. The ADC reference window specifies the full scalemeasurement range, such as −V_(ref) to +V_(ref) millivolts/volts, and isprogrammable on the ADC chip. During the sequencing phase of thenanopore based sequencing chip, the signal useful for nucleic acidsequencing is very small, and therefore the ADC reference window isprogrammed to a small voltage window, such that the small signal may beresolved into different discrete levels. However the |ΔV_(ADC)| inresponse to a phase change of the square wave V_(liq) for a cell with aruptured lipid bilayer is considerably larger than the signal useful fornucleic acid sequencing. As a result, a small ADC reference window thatis suitable for nucleic acid sequencing may likely cause the ΔV_(ADC)signal read by the ADC to saturate. Lowering the V_(liq) magnitude toprevent the saturation of the ΔV_(ADC) signal is not a viable solutionbecause the V_(liq) magnitude is constrained by the biochemistry fornucleic acid sequencing. Consequently, the diagnostic techniquedescribed above may no longer reliably distinguish between an intactlipid bilayer and a ruptured one in a cell, especially when themodulation frequency of V_(liq) is beyond a certain threshold level,e.g., 80 Hz or 100 Hz. Therefore, improved techniques to detect a stateof a lipid membrane in a cell of a nanopore based sequencing chip duringdifferent phases of the system would be desirable.

FIG. 12 illustrates an exemplary plot of an improved V_(liq) waveform1200 for detecting a state of a lipid membrane in a cell of a nanoporebased sequencing chip during different phases (including the sequencingphase) of the system. The states include a lipid membrane with multiplelayers of lipid molecules and solvent combined together, a lipidbilayer, and a ruptured lipid bilayer. The improved V_(liq) waveform1200 may be applied to circuitry 600 in FIG. 6A. Unlike the originalV_(liq) waveform, which is a square wave that switches between twomagnitudes when the waveform changes from a positive phase to a negativephase, or vice versa (e.g., see FIG. 11B), the improved V_(liq) waveform1200 includes an additional monitoring signal 1202. In FIG. 12 , thex-axis plots the frames and the y-axis plots the V_(liq) waveform inunits of mV. In this particular embodiment, the additional monitoringsignal 1202 is inserted when V_(liq) waveform 1200 switches from abright period to a dark period. In particular, V_(liq) switches from thebright magnitude (80 mV at frame #4 or #15) to an intermediatemonitoring signal magnitude (60 mV at frame #5 or #16) before itswitches to the dark magnitude (10 mV at frame #6 or #17). Anintermediate change in the V_(liq) AC voltage is inserted between twomagnitudes of the AC voltage, the bright magnitude and the darkmagnitude. In some other embodiments, the additional monitoring signalis inserted when the waveform switches from a dark period to a brightperiod. In these embodiments, V_(liq) switches from the dark magnitudelevel to an intermediate monitoring signal magnitude before it switchesto the bright magnitude level. The intermediate monitoring signalmagnitude may be a small delta in voltage (e.g., −20 mV, as shown inFIG. 12 ) from the voltage level prior to the monitoring signal. Anintermediate change in the V_(liq) AC voltage is inserted between twomagnitudes of the AC voltage, the dark magnitude and the brightmagnitude. In some embodiments, the delta change in voltage may be afraction (e.g., 2/7, as shown in FIG. 12 ) of the total change inmagnitude of V_(liq) when the waveform switches between phases. In FIG.12 , the magnitude change of V_(liq) waveform 1200 from a bright periodto a dark period is 70 mV. In other embodiments, the magnitude range maybe smaller or larger, e.g., from 50 mV to 500 mV, depending on thebiochemistry for nucleic acid sequencing. The sampling or frame rate mayrange from 1-10 kHz.

FIG. 13 illustrates that to detect a cell with a short-circuit conditionduring the sequencing phase, circuitry 600 may monitor a delta voltagechange, ΔV_(ADC), at integrating capacitor 608 (neap) in response to adelta voltage change (ΔV_(liq)) applied to the bulk liquid in contactwith the lipid membrane/bilayer when V_(liq) switches from a brightmagnitude to a monitoring signal magnitude. In the top plot of FIG. 13 ,the x-axis plots the frames and the y-axis plots the V_(liq) waveform1300 in units of mV. In the bottom plot of FIG. 13 , the x-axis plotsthe ADC samples and the y-axis plots the ADC counts read by the ADC atintegrating capacitor 608. As shown in the top plot, a narrow ADCreference window 1310 (−25 mV to +25 mV) is programmed on the ADC for animproved resolution for the relatively small signal that is used fornucleic acid sequencing. The narrow ADC reference window 1310 causes theADC value 1306 and ADC value 1308 to saturate at 0 and 255 count,respectively. However, because the monitoring signal 1302 introduces avoltage change that is only a fraction of the total change in magnitudeof V_(liq) when the waveform switches between phases, the response tothe monitoring signal 1304 does not saturate and the corresponding deltavoltage change may be used to more reliably detect whether the lipidbilayer is intact or ruptured. For example, two threshold levels may beused to determine the state of a lipid membrane, wherethreshold1<threshold2. If (|ΔV_(ADC)|<threshold)), then it is determinedthat the lipid membrane has multiple layers of lipid molecules andsolvent combined together, and is not yet a lipid bilayer. If((threshold1<=|ΔV_(ADC)|<threshold2), then it is determined that a lipidbilayer is intact. If (|ΔV_(ADC)|>=threshold2), then it is determinedthat a lipid bilayer is ruptured.

As shown in FIG. 13 , the delta change in voltage introduced bymonitoring signal 1302 is about ½ of the total change in magnitude ofV_(liq) when the waveform switches between phases. However, depending onthe ADC reference window selected, the monitoring signal magnitude maybe scaled to any appropriate fraction of the total change in magnitudeof V_(liq) when the V_(liq) waveform switches between phases, providedthat the response to the monitoring signal remains unsaturated.

In some embodiments of the nanopore based sequencing chip, the nanoporearray is divided into cell banks. For example, each cell bank mayinclude M rows×N columns of cells. Row and column select lines are usedto control the states of the individual cells. M and N may be anyinteger numbers. For example, a cell bank that is 8 k in size (referredto as a bank8 k) may be configured as 64 rows by 128 columns, with atotal of 64×128 cells. Since each cell bank is autonomous, the nanoporearray may be scaled by adding additional banks. For example, a 128 karray may be implemented as sixteen bank8 k elements. A 512 k array maybe implemented as an 8×8 array of bank8 k elements. In some embodiments,the nanopore array may be scaled to include millions of cells.

In some embodiments, the cells in any given row of a cell bank share thesame integration time interval, i.e., each of the cells in the same rowstarts and ends its integration at the same time, and the integrationtime intervals of adjacent rows are staggered from one another. Becausethe integration time intervals of adjacent rows are offset in time fromeach other, the detection technique using the monitoring signaldescribed above has a row dependence effect, and the overall performanceof the detection technique is degraded as a result. As will be describedin greater detail below, the row dependence effect may be mitigated byintroducing a global pre-charge signal for synchronizing the integrationof the cells in different rows of the cell bank when V_(liq) is about toswitch to the monitoring signal, thereby improving the overallperformance of the detection technique.

FIG. 14 illustrates that the cells in any given row of a cell bank sharethe same integration time intervals, but that the integration timeintervals of adjacent rows are staggered from each other, causing a rowdependence effect which degrades the overall performance of thedetection of states of lipid membranes. The top part of FIG. 14 showsthe zoomed-in version of V_(liq) waveform 1300 (see FIG. 13 ). V_(liq)1300 starts at a bright magnitude 1422 and switches to an intermediatemonitoring signal magnitude 1424 before switching to a dark magnitude1426.

The bottom part of FIG. 14 shows a timing diagram that indicates theframes, the integration time intervals for different rows (e.g., row 0,row 1, and row 63) of the cell bank, and the readout timing of the ADCs.

The vertical arrows 1401-1405 define the beginning of the frames ofV_(liq) waveform 1300; they are the time instances when the system maydetermine whether to maintain the V_(liq) magnitude at its current valueor switch to a different value. At frames 1401 and 1402, V_(liq) 1300 ismaintained at bright magnitude 1422. At frame 1403, V_(liq) 1300switches to intermediate monitoring signal magnitude 1424. At frame1404, V_(liq) 1300 switches to dark magnitude 1426. And at frame 1405,V_(liq) 1300 is maintained at dark magnitude 1426.

Each of the horizontal arrows indicates the integration time interval ofthe cells in a particular row of the cell bank. For example, the top setof horizontal arrows indicate the integration time intervals of thecells in row 0, the second set of horizontal arrows indicate theintegration time intervals of the cells in row 1, and the bottom set ofhorizontal arrows indicate the integration time intervals of the cellsin row 63. As shown in the figure, the integration time intervals ofadjacent rows are offset in time from each other. For example, thebeginning of an integration time interval for row 0 and the beginning ofan integration time interval for row 1 are offset by a small delta timedifference; the beginning of an integration time interval for row 1 andthe beginning of an integration time interval for row 2 are offset by asmall delta time difference, and so on.

The cells in any given row of a cell bank share the same integrationtime interval, but the integration time intervals of adjacent rows arestaggered from each other because the timing of the reset signal 603used to trigger the pre-charging of the integrating capacitors 608 (seeFIG. 6A) for cells in one row is different from those for other rows.

For example, at time 1417, each of the cells in row 0 has its resetsignal 603 closing the cell's switch 601, such that its integratingcapacitor 608 is connected to voltage source V_(pre) 605. Afterintegrating capacitor 608 is pre-charged, reset signal 603 is used toopen switch 601 such that integrating capacitor 608 is disconnected fromvoltage source V_(pre) 605. At this point, integrating capacitor 608starts to discharge and the integration time interval 1411 for row 0begins. After the integration time interval 1411 for row 0 is over, thevoltage stored in integrating capacitor 608 of each of the cells in row0 may be read out by ADC 610 at time 1414. After the sampling by ADC610, integrating capacitor 608 is pre-charged again by using resetsignal 603 to close switch 601, such that integrating capacitor 608 isconnected to voltage source V_(pre) 605 again. The steps of pre-chargingthe integrating capacitor 608, waiting a fixed period of time for theintegrating capacitor 608 to integrate, and sampling the voltage storedin integrating capacitor 608 by ADC 610 are then repeated in cycles.

The reset signal 603 used to trigger the pre-charging and integration ofthe cells in row 1 lags behind that of row 0 by a small delta timedifference, at time 1418. After the integration time interval 1412 forrow 1 is over, the voltage stored in integrating capacitor 608 of eachof the cells in row 1 may be read out by ADC 610 at time 1415.Similarly, the reset signal 603 used to trigger the pre-charging andintegration of the cells in row 2 lags behind that of row 1 by a smalldelta time difference, the reset signal 603 of the cells in row 3 lagsbehind that of row 2 by a small delta time difference, and so on. Thispattern is repeated until row 63. In particular, at time 1419, each ofthe cells in row 63 has its reset signal 603 triggering the pre-chargingand integration of the cells. After the integration time interval 1413for row 63 is over, the voltage stored in integrating capacitor 608 ofeach of the cells in row 63 is read out by ADC 610 at time 1416.

Because the integration time intervals of different rows of cells areoffset in time, when V_(liq) 1300 switches from bright magnitude 1422 tointermediate monitoring signal magnitude 1424, the cells in twodifferent rows experience the monitoring voltage change at differenttimes within their respective integration time intervals, therebycausing the ADC outputs of the two different rows to differ. Forexample, the cells in row 0 experience the monitoring voltage change atthe latter part of integration time interval 1411, while the cells inrow 63 experience the monitoring voltage change at the earlier part ofintegration time interval 1413; therefore, the ADC outputs in row 0versus those in row 63 have greater variations than the ADC outputs inthe same row, thereby causing the row dependence effect.

FIG. 15 illustrates that a global pre-charge signal 1502 is used tosynchronize the integration of the cells in different rows of the cellbank when V_(liq) is about to switch to the intermediate monitoringsignal. FIG. 14 and FIG. 15 are similar apart from a few differences:one difference is the introduction of the global pre-charge signal 1502,as will be described in greater detail below; another difference is thatin FIG. 15 , the integration time intervals of adjacent rows are nolonger staggered from each other the way they are in FIG. 14 (seeintegration time intervals 1511, 1512, and 1513) when V_(liq) 1300 isswitched to intermediate monitoring signal magnitude 1424. For example,the beginning of integration time interval 1511 for row 0, the beginningof integration time interval 1512 for row 1, and the beginning ofintegration time interval 1513 for row 63 now occur at the same time.

Global pre-charge signal 1502 is a pre-charge signal that is used tocontrol switch 601 across all of the cells in a cell bank. In contrast,reset signal 603 is a pre-charge signal that is used to control switch601 in a single cell or only cells in a single row of the cell bank. Asshown in FIG. 15 , global pre-charge signal 1502 is set to high at frame1402 (the frame before V_(liq) 1300 is switched to intermediatemonitoring magnitude 1424), which closes all the switches 601 in thecells of the cell bank, such that all the integrating capacitors 608 areconnected to voltage source V_(pre) 605 and pre-charged to the V_(pre)voltage level. Global pre-charge signal 1502 is then set to low at frame1403 (the frame when V_(liq) 1300 is switched to intermediate monitoringmagnitude 1424), which opens all the switches 601 in the cells of thecell bank, such that all integrating capacitors 608 are disconnectedfrom voltage source V_(pre) 605 and start to discharge and integrate atthe same time (see integration time intervals 1511, 1512, and 1513).

After the integration time interval 1511 for row 0 is over, the voltagestored in integrating capacitor 608 of each of the cells in row 0 may beread out by ADC 610 at time 1514. After row 0 is read, the integrationtime interval 1512 for row 1 is over, and the voltage stored inintegrating capacitor 608 of each of the cells in row 1 may be read outby ADC 610 at time 1515. Similarly, the subsequent rows are read out oneby one, until row 63 is read out at time 1516.

Although the length of the integration time intervals for different rowsare not identical (for example, the lengths of 1511, 1512, and 1513 aredifferent) when V_(liq) 1300 is at intermediate monitoring signalmagnitude 1424, all of the cells—irrespective of which row they belongto—integrate while the V_(liq) magnitude is maintained at intermediatemonitoring signal magnitude 1424. As a result, the ADC outputs indifferent rows have less variance, thereby reducing the row dependenceeffect.

However, one drawback of using the global pre-charge signal 1502 is thatone set of sequencing data is lost when global pre-charge signal 1502 isset high between 1402 and 1403. Data points 1507 are invalid because thevoltage stored in integrating capacitor 608 of all the cells in the cellbank are pre-charged to the V_(pre) voltage level.

FIG. 16 illustrates that a modified global pre-charge signal 1602 isused to synchronize the integration of the cells in different rows ofthe cell bank when V_(liq) is about to switch to the intermediatemonitoring signal. FIG. 15 and FIG. 16 are similar apart from a fewdifferences: one difference is the modification of the global pre-chargesignal 1602, as will be described in greater detail below; anotherdifference is that in FIG. 16 , the data points 1607 that are useful forsequencing purposes are no longer corrupted by the global pre-chargesignal 1602.

Global pre-charge signal 1602 is a pre-charge signal that is used tocontrol switch 601 across all of the cells in a cell bank. In contrast,reset signal 603 is a pre-charge signal that is used to control switch601 in a single cell or only cells in a single row of the cell bank. Asshown in FIG. 16 , global pre-charge signal 1602 is set to high at time1621, which closes all the switches 601 in the cells of the cell banksuch that all the integrating capacitors 608 are connected to voltagesource V_(pre) 605 and pre-charged to the V_(pre) voltage level. Globalpre-charge signal 1602 is then set to low at time 1620, which opens allthe switches 601 in the cells of the cell bank such that all integratingcapacitors 608 are disconnected from voltage source V_(pre) 605 andstart to discharge and integrate at the same time (see integration timeintervals 1611, 1612, and 1613).

After the integration time interval 1611 for row 0 is over, the voltagestored in integrating capacitor 608 of each of the cells in row 0 may beread out by ADC 610 at time 1614. After row 0 is read, the integrationtime interval 1612 for row 1 is over, and the voltage stored inintegrating capacitor 608 of each of the cells in row 1 may be read outby ADC 610 at time 1615. Similarly, the subsequent rows are read out oneby one, until row 63 is read out at time 1616.

Although the length of the integration time intervals for different rowsare not identical (for example, the lengths of 1611, 1612, and 1613 aredifferent), when V_(liq) 1300 is at intermediate monitoring signalmagnitude 1424, all of the cells—irrespective of which row they belongto—integrate while the V_(liq) magnitude is maintained at intermediatemonitoring signal magnitude 1424. As a result, the ADC outputs indifferent rows have less variance, thereby reducing the row dependenceeffect.

In addition, the data points 1607 that are useful for sequencingpurposes are no longer corrupted by global pre-charge signal 1602.Global pre-charge signal 1602 is set to high at time 1621, which occursafter time 1622, the time when the readout during frame 1402 (the framethat is prior to the monitoring voltage step) has been completed,thereby preserving the data points 1607 that are useful for sequencingpurposes. Global pre-charge signal 1602 is then set to low at time 1620.In some embodiments, time 1620 is at the same time as frame 1403, theframe when V_(liq) 1300 is switched to intermediate monitoring magnitude1424. In some embodiments, time 1620 is substantially at the same timeas frame 1403, either immediately prior to frame 1403 or immediatelyafter frame 1403.

FIG. 17A illustrates the row dependence effect of the lipid membranestate detection technique. In FIG. 17A, the bottom portion is a plot ofthe observed voltage change ΔV_(ADC) in response to the monitoringsignal in cells that belong to different rows of a cell bank whenruptured lipid bilayers are detected. On the x-axis, row numbers 0-63corresponds to row numbers 0-63 in cell bank 1, row numbers 64-127corresponds to row numbers 0-63 in cell bank 2, row numbers 128-191corresponds to row numbers 0-63 in cell bank 3, and row numbers 192-255corresponds to row numbers 0-63 in cell bank 4. On the y-axis, theresponses to the monitoring signal are plotted in ADC counts. As shownin the bottom portion of FIG. 17A, the response signals graduallyincrease as the row number increases. The majority of the responsesignals range from around 15 to 40 ADC counts. The top portion of FIG.17A is a histogram that shows the distribution of the response signals.The response signals range from 0 to about 40 ADC counts.

FIG. 17B illustrates that the row dependence effect is significantlyreduced by using the global pre-charge signal in FIG. 16 . In FIG. 17B,the bottom portion is a plot of the observed voltage change ΔV_(ADC) inresponse to the monitoring signal in cells that belong to different rowsof a cell bank when ruptured lipid bilayers are detected. On the x-axis,row numbers 0-63 corresponds to row numbers 0-63 in cell bank 1, rownumbers 64-127 corresponds to row numbers 0-63 in cell bank 2, rownumbers 128-191 corresponds to row numbers 0-63 in cell bank 3, and rownumbers 192-255 corresponds to row numbers 0-63 in cell bank 4. On they-axis, the responses to the monitoring signal are plotted in ADCcounts. As shown in the bottom portion of FIG. 17B, the response signalsincrease only slightly as the row number increases. The majority of theresponse signals range from around 40 to 60 ADC counts. The top portionof FIG. 17B is a histogram that shows the distribution of the responsesignals. The response signals range from 40 to about 60 ADC counts, withapproximately 50 ADC counts being the most likely ADC value.

FIG. 18A is a plot of the observed voltage change ΔV_(ADC) in responseto the monitoring signal in cells that belong to different rows of acell bank when ruptured lipid bilayers are detected. FIG. 18B is ahistogram that shows the distribution of the response signals whenruptured lipid bilayers are detected. FIG. 18C is a plot of the observedvoltage change ΔV_(ADC) in response to the monitoring signal in cellsthat belong to different rows of a cell bank when ruptured lipidbilayers are not detected. FIG. 18D is a histogram that shows thedistribution of the response signals when ruptured lipid bilayers arenot detected.

As shown in FIG. 18D, the response signals range from 0 to about 45 ADCcounts when ruptured lipid bilayers are not detected; and as shown inFIG. 18B, the response signals range from about 55 to 120 ADC countswhen ruptured lipid bilayers are detected. Since the response signalsrange for the two lipid bilayer conditions do not overlap with eachother, the detection technique may be used to reliably determine whethera lipid bilayer is intact or ruptured.

The above disclosed detection technique has many advantages. Theselection of the monitoring signal magnitude is decoupled from theselection of the V_(liq) bright/dark magnitudes. The ADC referencewindow may be selected to increase the resolution of the signals usedfor nucleic acid sequencing, without causing the response signals to themonitoring signal to saturate. The technique may be used to detect thecondition of bilayers even during the sequencing stage. In addition, thetechnique may reliably detect the condition of bilayers when themodulation frequency of V_(liq) is beyond 100 Hz.

As described above, when the lipid solvent mixture is first depositedinto the cells to form the lipid bilayers, some of the cells have lipidbilayers spontaneously formed, but some of the cells merely have a thicklipid membrane with multiple layers of lipid molecules combined with thesolvent spanning across each of the wells of the cells. In order toincrease the yield of the nanopore based sequencing chip (i.e., thepercentage of cells in the nanopore based sequencing chip with properlyformed lipid bilayers and nanopores), the nanopore based sequencing chipmay perform additional steps to facilitate the formation of lipidbilayers in additional cells. For example, applying an electricallipid-thinning stimulus to the cells that have not had lipid bilayersformed therein yet can improve the efficiency of liquid flow above thethick lipid membranes, thereby facilitating the removal of any excesslipid solvent such that the thick lipid membranes can be thinned out andtransitioned into lipid bilayers more efficiently. Applying theelectrical lipid-thinning stimulus to the cells that have not had lipidbilayers formed therein yet will also create electrostatic forces thattend to squeeze out the excess lipid solvent and thin out the thicklipid membranes into lipid bilayers. On the other hand, the cells thathave already had lipid bilayers properly formed therein should not befurther exposed to the same electrical lipid-thinning stimulus, as theelectrical stimulus may cause some of the thin lipid bilayers to breakdown. Therefore, it is advantageous to use the non-destructive techniquedescribed in the present application to detect and separate the portionof the cells in the nanopore based sequencing chip that have lipidbilayers formed therein from the portion of the cells that do not havelipid bilayer properly formed therein yet. By dividing the cells intodifferent groups, the cells in different groups can be processeddifferently, thereby achieving greater efficiency and increasing theoverall yield of the nanopore based sequencing chip.

FIG. 19 illustrates an embodiment of a process 1900 for an improvedtechnique of forming lipid layers in the cells of a nanopore basedsequencing chip. In some embodiments, the nanopore based sequencing chipof FIG. 19 includes a plurality of cells 100 of FIG. 1 . In someembodiments, the nanopore based sequencing chip of FIG. 19 includes aplurality of cells 500 of FIG. 5 . In some embodiments, the nanoporebased sequencing chip of FIG. 19 includes circuitries 600 of FIGS. 6Aand 6B.

Process 1900 includes steps in which different types of fluids (e.g.,liquids or gases) are flowed through the cells of the nanopore basedsequencing chip via a flow chamber. Multiple fluids with significantlydifferent properties (e.g., compressibility, hydrophobicity, andviscosity) are flowed over an array of sensors on the surface of thenanopore based sequencing chip. For improved efficiency, each of thesensors in the array should be exposed to the fluids in a consistentmanner. For example, each of the different types of fluids should beflowed over the nanopore based sequencing chip such that the fluid maybe delivered to the chip, evenly coating and contacting each of thecells' surfaces, and then delivered out of the chip. As described above,a nanopore based sequencing chip incorporates a large number of sensorcells configured as an array. As the nanopore based sequencing chip isscaled to include more and more cells, achieving an even flow of thedifferent types of fluids across the cells of the chip becomes morechallenging.

In some embodiments, the nanopore based sequencing system that performsprocess 1900 of FIG. 19 includes an improved flow chamber having aserpentine fluid flow channel that directs the fluids to traverse overdifferent sensors of the chip along the length of the channel. FIG. 20illustrates the top view of a nanopore based sequencing system 2000 withan improved flow chamber enclosing a silicon chip that allows liquidsand gases to pass over and contact sensors on the chip surface. The flowchamber includes a serpentine or winding flow channel 2008 that directsthe fluids to flow directly above a single column (or a single row) ofsensor banks 2006 (each bank including several thousands of sensorcells) from one end of the chip to the opposite end and then directs thefluids to repeatedly loop back and flow directly above other adjacentcolumns of sensor banks, until all of the sensor banks have beentraversed at least once. As shown in FIG. 20 , system 2000 includes aninlet 2002 and an outlet 2004.

With reference to FIG. 20 , a fluid is directed into system 2000 throughinlet 2002. Inlet 2002 may be a tube or a needle. For example, the tubeor needle may have a diameter of one millimeter. Instead of feeding theliquid or gas directly into a wide flow chamber with a single continuousspace, inlet 2002 feeds the liquid or gas into a serpentine flow channel2008 that directs the liquid or gas to flow directly above a singlecolumn of sensor banks 2006. The serpentine channel 2008 may be formedby stacking together a top plate and a gasket with dividers 2010 thatdivide the chamber into the serpentine channel to form a flow cell, andthen mounting the flow cell on top of the chip. Once the liquid or gasflows through the serpentine channel 2008, the liquid or gas is directedup through outlet 2004 and out of system 2000.

System 2000 allows the fluids to flow more evenly on top of all thesensors on the chip surface. The channel width is configured to benarrow enough such that capillary action has an effect. Moreparticularly, the surface tension (which is caused by cohesion withinthe fluid) and adhesive forces between the fluid and the enclosingsurfaces act to hold the fluid together, thereby preventing the fluid orthe air bubbles from breaking up and creating dead zones. For example,the channel may have a width of 1 millimeter or less. The narrow channelenables controlled flow of the fluids and minimizes the amount ofremnants from a previous flow of fluids or gases.

With reference to FIG. 19 , at 1902, a salt/electrolyte buffer solutionis flowed through the cells of the nanopore based sequencing chip viathe flow chamber to substantially fill the wells in the cells with thesalt buffer solution. The salt buffer solution may include one of thefollowing: lithium chloride (LiCl), sodelectium chloride (NaCl),potassium chloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂)), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂). In some embodiments, theconcentration of the salt buffer solution is 300 mM (millimolar).

At 1904, a lipid and solvent mixture is flowed through the cells of thenanopore based sequencing chip via the flow chamber. In someembodiments, the lipid and solvent mixture includes lipid molecules suchas diphytanoylphosphatidylcholine (DPhPC). In some embodiments, thelipid and solvent mixture includes decane or tridecane. When the lipidand solvent mixture is first deposited into the cells to form the lipidbilayers, some of the cells have lipid bilayers spontaneously formed,but some of the cells merely have a thick lipid membrane (with multiplelayers of lipid molecules and solvent combined together) spanning acrosseach of the wells of the cells. In order to increase the yield of thenanopore based sequencing chip (i.e., the percentage of cells in thenanopore based sequencing chip with properly formed lipid bilayers andnanopores), the nanopore based sequencing chip will repeatedly gothrough two phases, an electrical lipid-thinning stimulus phase and abuffer flowing phase, to facilitate the formation of lipid bilayers inadditional cells.

The electrical lipid-thinning stimulus phase of process 1900 includessteps 1906, 1908, and 1910. In some embodiments, during this phase steps1906, 1908, and 1910 may be performed in the order as shown in FIG. 19 .In some embodiments, steps 1906, 1908, and 1910 may be performed in adifferent order. In some embodiments, the steps may be performedsimultaneously.

At 1906, the non-destructive technique described in the presentapplication is used to detect whether a lipid bilayer is formed in acell using circuitry 600 of FIG. 6A and FIG. 6B. The detection includesmonitoring a voltage change, ΔV_(ADC), at integrating capacitor 608(neap) in response to a voltage change (ΔV_(liq)) applied to the bulkliquid in contact with the lipid membrane/bilayer. Cells that have lipidbilayers detected are separated into a different group from the cellsthat do not have lipid bilayers detected. Within each of the cells withlipid bilayers detected, pass device 606 is opened in order todisconnect the lipid bilayer and the electrodes from the measurementcircuitry 600, such that the electrical lipid-thinning stimulus isdisabled from being applied to the cell.

At 1908, an electrical lipid-thinning stimulus is applied to the cellsof the nanopore based sequencing chip. Applying the electricallipid-thinning stimulus to the cells that have not had lipid bilayersformed therein yet can improve the efficiency of liquid flow above thethick lipid membranes, thereby facilitating the removal of any excesslipid solvent such that the thick lipid membranes can be thinned out andtransitioned into lipid bilayers more efficiently. Applying theelectrical lipid-thinning stimulus to the cells that have not had lipidbilayers formed therein yet will also create electrostatic forces thattend to squeeze out the excess lipid solvent and thin out the thicklipid membranes into lipid bilayers. In some embodiments, the samecircuitry 600 of FIG. 6A and FIG. 6B may be used to apply the electricallipid-thinning stimulus. The only difference in the setup of circuitry600 between lipid bilayer detection and lipid thinning is that theabsolute magnitude of V_(liq) is lower for lipid bilayer detection. Forexample, the absolute magnitude V_(liq) for lipid bilayer detection maybe between 100 mV to 250 mV, while the absolute magnitude V_(liq) forlipid thinning may be between 250 mV to 500 mV.

At step 1910, it is determined whether the electrical lipid-thinningstimulus phase is finished. In some embodiments, the electricallipid-thinning stimulus is applied to any cells that have not beendetected as having lipid bilayers therein for a period of two seconds.However, other predetermined period of time may be used as well. If thephase is not over yet, then process 1900 returns to steps 1906 and 1908again until the time period is finished; otherwise, process 1900proceeds to the salt buffer solution flowing phase next.

The salt buffer solution flowing phase of process 1900 includes steps1912, 1914, and 1916. In some embodiments, during this phase steps 1912,1914, and 1916 may be performed in the order as shown in FIG. 19 . Insome embodiments, steps 1912, 1914, and 1916 may be performed in adifferent order. In some embodiments, the steps may be performedsimultaneously.

At 1912, the same non-destructive technique used at step 1906 is used todetect whether a lipid bilayer is formed in the cell using circuitry 600of FIG. 6A and FIG. 6B. Cells that have lipid bilayers detected areseparated into a different group from the cells that do not have lipidbilayers detected. Within each of the cells with lipid bilayersdetected, pass device 606 is opened in order to disconnect the lipidbilayer and the electrodes from the measurement circuitry 600.

At 1914, a salt/electrolyte buffer solution is flowed through the cellsof the nanopore based sequencing chip via the flow chamber. The purposeof flowing the salt buffer solution over the cells is to facilitate theformation of a lipid bilayer over each of the cells. When the saltbuffer solution is flowed over the cells, the thickness of the lipid andsolvent mixture deposited on the cell is reduced, facilitating theformation of the lipid bilayer.

At 1916, it is determined whether the salt buffer solution flowing phaseis over. In some embodiments, salt buffer solution is flowed for aperiod of two seconds. However, other predetermined period of time maybe used as well. If the phase is not over yet, then process 1900 returnsto steps 1912 and 1914 again until the time period is finished;otherwise, process 1900 proceeds to step 1918.

At 1918, it is determined whether the electrical lipid-thinning stimulusphase and salt buffer solution flowing phase of process 1900 should berepeated. Different criteria may be used at this step. In someembodiments, the electrical lipid-thinning stimulus phase and saltbuffer solution flowing phase are performed a predetermined number oftimes. In some embodiments, the two phases are repeated until a targetyield for the nanopore based sequencing chip has been reached. In someembodiments, if the incremental number or percentage of cells that havejust been detected as having lipid bilayers formed during the last roundof thinning by the stimulus and the buffer solution flow is lower than apredetermined threshold, then process 1000 is terminated. In someembodiments, the two phases are repeated until the most recently appliedelectrical lipid-thinning stimulus level has reached a predeterminedmaximum threshold, e.g. 500 mV.

Process 1900 proceeds to step 1920 if the electrical lipid-thinningstimulus phase and salt buffer solution flowing phase of process 1900are going to be repeated next. At step 1920, the next electricallipid-thinning stimulus to be applied is determined. In someembodiments, the electrical lipid-thinning stimulus level is increasedby a fixed predetermined amount, e.g., an increment of 100 mV. In someembodiments, if the incremental number or percentage of cells that havejust been detected as having lipid bilayers formed during the lastiteration is lower than a predetermined threshold, then the electricallipid-thinning stimulus level is increased by a fixed predeterminedamount; otherwise, the previous electrical lipid-thinning stimulus isfound to be effective and thus the same electrical lipid-thinningstimulus level is used again.

FIGS. 21A, 21B, and 21C are histograms that illustrate that as theelectrical lipid-thinning stimulus phase and salt buffer solutionflowing phase of process 1900 repeats a number of times, the overallpercentage of cells in the nanopore based sequencing chip with properlyformed lipid bilayers (i.e., the yield of the nanopore based sequencingchip) increases. For each of the figures, the x-axis is the voltagechange at integrating capacitor 608(n _(cap)), ΔV_(ADC), in response toa voltage change (ΔV_(liq)) applied to the bulk liquid in contact withthe lipid membrane/bilayer, while the y-axis is the number of cells withits ΔV_(ADC) value within certain ΔV_(ADC) bins. FIG. 21A illustratesthe initial distribution of cells with different ΔV_(ADC) values. FIG.21B illustrates the distribution of cells with different ΔV_(ADC) valuesafter the lipid-thinning stimulus phase and salt buffer solution flowingphase of process 1000 have repeated a number of times. FIG. 21Cillustrates the distribution of cells with different ΔV_(ADC) valuesafter the lipid-thinning stimulus phase and salt buffer solution flowingphase of process 1000 have repeated an even greater number of times. Inthis example, cells that have a ΔV_(ADC) value of 50 or above aredetermined as having lipid bilayers formed therein. As shown in FIG.21A, initially, only a small number of cells have lipid bilayersdetected. As shown in FIG. 21B, after the lipid-thinning stimulus phaseand salt buffer solution flowing phase of process 1900 have repeated anumber of times, the number of cells having lipid bilayers detectedincreases. Finally, as shown in FIG. 21C, after the lipid-thinningstimulus phase and salt buffer solution flowing phase of process 1900have repeated an even greater number of times, a high majority of thecells in the nanopore based sequencing chip has lipid bilayers detected.

FIG. 22 illustrates an embodiment of a timing diagram for a bilayermeasurement phase, an electrical lipid-thinning stimulus phase, and asalt buffer solution flowing phase. In this example, after lipids aredeposited into the cells, the bilayer measurement phase is started. Thebilayer measurement phase lasts about 2 seconds in time. During thisphase, all of the cells are enabled. The absolute value of V_(liq) is250 mV.

The bilayer measurement phase is followed by the electricallipid-thinning stimulus phase, which lasts about 2 seconds in time.During this phase, when the absolute value of ΔV_(ADC) (ΔV_(ADC) I)exceeds threshold1 within a cell, then a lipid bilayer is detectedwithin the cell and the cell is disconnected from the voltage source.The absolute value of V_(liq) is between 250-500 mV.

The electrical lipid-thinning stimulus phase is followed by the saltbuffer solution flowing phase, and the latter lasts about 2-10 secondsin time. During this phase, when the absolute value of ΔV_(ADC)(ΔV_(ADC) I) exceeds threshold2 within a cell, then a lipid bilayer isdetected within the cell and the cell is disconnected from the voltagesource. The absolute value of V_(liq) is 250 mV.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

What is claimed is:
 1. A method of detecting a state of a membrane in acell of a nanopore based sequencing chip, comprising: applying analternating current (AC) voltage across a membrane disposed between aworking electrode and a counter electrode, the membrane electricallycoupled with a capacitor, wherein the AC voltage alternates between afirst voltage and a second voltage; periodically sampling a voltageacross the capacitor; determining a change in the sampled voltage acrossthe capacitor in response to a monitoring signal, wherein the monitoringsignal is a change in the AC voltage with a magnitude that is less thanthe difference between the first voltage and the second voltage; andcomparing the change in the sampled voltage across the capacitor againsta predetermined threshold, and determining the state of the membranebased on the comparisons against the predetermined threshold, whereinthe state of the membrane is classified as a membrane with no more thantwo molecular layers when the change in sampled voltage is greater thanthe predetermined threshold.
 2. The method of claim 1, wherein themembrane with no more than two molecular layers is a lipid bilayer.
 3. Amethod of detecting a state of a membrane in a cell of a nanopore basedsequencing chip, comprising: applying an alternating current (AC)voltage across a membrane disposed between a working electrode and acounter electrode, the membrane electrically coupled with a capacitor,wherein the AC voltage alternates between a first voltage and a secondvoltage; periodically sampling a voltage across the capacitor;determining a change in the sampled voltage across the capacitor inresponse to a monitoring signal, wherein the monitoring signal is achange in the AC voltage with a magnitude that is less than thedifference between the first voltage and the second voltage; andcomparing the change in the sampled voltage across the capacitor againsta first predetermined threshold and a second predetermined threshold,wherein the first predetermined threshold is less than the secondpredetermined threshold, and determining the state of the membrane basedon the comparisons against the first predetermined threshold and thesecond predetermined threshold, wherein the state of the membrane isclassified as a membrane with no more than two molecule layers when thechange in sampled voltage is greater than or equal to the firstpredetermined threshold and less than the second predeterminedthreshold.
 4. The method of claim 3, wherein the membrane is classifiedas a membrane with more than two molecule layers when the change insampled voltage is less than the first predetermined threshold.
 5. Themethod of claim 3, wherein the membrane is classified as a rupturedmembrane when the change in sampled voltage is greater than or equal tothe second predetermined threshold.
 6. The method of claim 5, furthercomprising: in response to the determination that the membrane hasruptured, disabling further electrical stimuli from being applied acrossthe membrane by opening a switch in the cell.